title: A robust immunoassay for pregnancy-associated plasma protein-A2 based on analysis of circulating antigen: establishment of normal ranges in pregnancy.
abstract: Pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, two homologous metzincin metalloproteases, are both tightly linked to regulation within the insulin-like growth factor (IGF) system because of their specific cleavage of IGF binding proteins. Recent studies suggest that PAPP-A may be involved in clinical conditions related to unwanted cellular growth, and the circulating levels of PAPP-A is an established biomarker in prenatal screening for chromosomal abnormalities. Microarray data indicate that PAPP-A2 has potential as a biomarker for pre-eclampsia. However, well-characterized immunological methods of quantification are not available. We therefore developed monoclonal antibodies against recombinant PAPP-A2. The antibodies were epitope mapped against recombinantly expressed chimeras between PAPP-A2 and PAPP-A. Furthermore, circulating PAPP-A2 was immunoaffinity purified and characterized by sequence analysis and mass spectrometry. Unlike PAPP-A, PAPP-A2 is a noncovalent dimer in which each subunit of 1558 amino acids originates from all of the 22 predicted coding exons. A previously hypothesized variant (PAPP-E) does not exist, but low amounts of a C-terminally truncated PAPP-A2 variant was detected. A sensitive and robust ELISA for full-length PAPP-A2 was developed and used to establish normal ranges of PAPP-A2 through pregnancy. The functional sensitivity of this ELISA at 20% CV was 0.08 ng/ml, and the serum concentration of PAPP-A2 was found to increase during pregnancy in agreement with placental synthesis. The existence of this assay will enable an assessment of the biomarker potential of PAPP-A2 in pre-eclampsia as well as other clinical conditions.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C DK-8000, Denmark.
authors.forename: S
authors.initials: S
authors.lastname: Kløverpris
authors.forename: E
authors.initials: E
authors.lastname: Gaidamauskas
authors.forename: L C V
authors.initials: LC
authors.lastname: Rasmussen
authors.forename: M T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: C
authors.initials: C
authors.lastname: Kronborg
authors.forename: U B
authors.initials: UB
authors.lastname: Knudsen
authors.forename: M
authors.initials: M
authors.lastname: Christiansen
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: C
authors.initials: C
authors.lastname: Oxvig
mesh_terms: D000818:Animals; D000941:Antigens / Q000097:blood; D015415:Biomarkers / Q000097:blood; D005260:Female; D005315:Fetal Diseases / Q000097:blood / Q000175:diagnosis; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods; D051379:Mice; D018345:Mice, Knockout; D011225:Pre-Eclampsia / Q000097:blood / Q000175:diagnosis; D011247:Pregnancy / Q000097:blood*; D011266:Pregnancy-Associated Plasma Protein-A / Q000032:analysis*; D011296:Prenatal Diagnosis / Q000379:methods* / Q000592:standards*; D012016:Reference Values; D012680:Sensitivity and Specificity
chemical_list: D000941:Antigens; D015415:Biomarkers; C430889:PAPPA2 protein, human; D011266:Pregnancy-Associated Plasma Protein-A
keywords: ELISA; PAPP-A2; biomarker; pappalysin-2; pregnancy